Methods and compositions involving immunostimulatory oligodeoxynucleotides

ABSTRACT

Described is an immunostimulatory oligodeoxynucleic acid molecule (ODN) having the structure according to formula (I), wherein any NMP is a 2′ deoxynucleoside monophosphate or monothiophosphate, selected from the group consisting of deoxyadenosine-, deoxyguanosine-, deoxyinosine-, deoxycytosine-, deoxyuridine-, deoxythymidine-, 2-methyl-deoxyinosine-, 5-methyl-deoxycytosine-, deoxypseudouridine-, deoxyribosepurine-, 2-amino-deoxyribosepurine-, -6-S-deoxyguanine-, 2-dimethyl-deoxyguanosine- or N-isopentenyl-deoxyadenosine-monophosphate or -monothiophosphate, NUC is a 2′ deoxynucleoside, selected from the group consisting of deoxyadenosine-, deoxyguanosine-, deoxyinosine-, deoxycytosine-, deoxyuridine-, deoxythymidine-, 2-methyl-deoxyinosine-, 5-methyl-deoxycytosine-, deoxypseudouridine-, deoxyribosepurine-, 2-amino-deoxyribosepurine-, 6-S-deoxyguanine-, 2-dimethyl-deoxyguanosine- or N-isopentenyl-deoxyadenosine, any X is O or S, a and b are integers from 0 to 100 with the proviso that a+b is between 4 and 150, B and E are common groups for 5′ or 3′ ends of nucleic acid molecules, as well as a pharmaceutical composition containing such ODNs.

This application is a continuation of U.S. patent application Ser. No.13/786,815, filed Mar. 6, 2013, now U.S. Pat. No. 8,945,591, which is acontinuation of U.S. patent application Ser. No. 10/297,555, filed Dec.6, 2002, now U.S. Pat. No. 8,568,742, which is a U.S. national phaseapplication under 35 U.S.C. §371 of PCT Application No. PCT/EP01/06433filed Jun. 7, 2001, which claims priority to Austrian Application Nos. A1973/2000 filed Nov. 23, 2000 and A 1000/2000 filed Jun. 8, 2000, theentire texts of which are specifically incorporated by reference hereinwithout disclaimer.

The present invention relates to immunostimulatory oligodeoxynucleicmolecules (ODNs) and pharmaceutical compositions containing such ODNs.

Vaccines can save more lives (and resources) than any other medicalintervention (Nossal, 1998). Owing to world-wide vaccination programsthe incidence of many fatal diseases has been decreased drastically.Although this notion is valid for a whole panel of diseases, e.g.tuberculosis, diphtheria, pertussis, measles and tetanus, there are noeffective vaccines for numerous infectious disease including most viralinfections, such as AIDS. There are also no effective vaccines for otherdiseases, infectious or non-infectious claiming millions the lives ofmillions of patients per year including malaria or cancer. In addition,the rapid emergence of antibiotic-resistant bacteria and microorganismscalls for alternative treatments with vaccines being a logical choice.Finally, the great need for vaccines is also illustrated by the factthat infectious diseases, rather than cardiovascular disorders or canceror injuries remain the largest cause of death and disability in theworld (Bloom and Widdus, 1998).

From an immunological point of view one major problem in the field ofvaccines today is that traditional vaccines (and/or theimmune-modulating compounds contained within these preparations) aredesigned to induce high levels of antibodies (Harrow and Lane, 1988).However, antibodies on their own are not effective in preventing a largenumber of diseases including most illnesses caused by viruses,intracellular bacteria, certain parasites and cancer. Examples for suchdiseases are, but are not restricted to, the above-mentioned HIV virusor Plasmodium spec. in case of malaria. In numerous experimental systemsit has been shown that the cellular arm of the immune system, includingT cells, rather than the humoral arm, is important for theseindications. Therefore, novel, innovative technologies are needed toovercome the limitations of conventional vaccines. The focus must be ontechnologies that reliably induce the cellular immune system, includingantigen specific T cells, which recognize molecules expressed onpathogen infected cells. Ideally, vaccines are designed that induce bothT cells distinguishing diseased and/or infected cells from normal cellsand, simultaneously, antibodies secreted by B cells recognisingpathogens in extracellular compartments.

Several established vaccines consist of live attenuated organism wherethe risk of reversion to the virulent wild-type strain exists. Inparticular in immunocompromised hosts this can be a live threateningscenario. Alternatively, vaccines are administered as a combination ofpathogen-derived antigens together with compounds that induce or enhanceimmune responses against these antigens (these compounds are commonlytermed adjuvant), since these subunit vaccines on their own aregenerally not effective.

Whilst there is no doubt that the above vaccines are valuable medicaltreatments, there is the disadvantage that, due to their complexity,severe side effects can be evoked, e.g. to antigens that are containedin the vaccine that display cross-reactivity with molecules expressed bycells of vaccinated individuals. In addition, existing requirements fromregulatory authorities, e.g. the World Health Organization (WHO), theFood and Drug Administration (FDA), and their European counterparts, forexact specification of vaccine composition and mechanisms of inductionof immunity, are difficult to meet.

Antigen presenting cells belong to the innate immune system, which hasevolved as a first line host defence that limits infection early afterexposure to microorganisms (Hoffmann et al., 1999). Cells of the innateimmune system recognize patterns or relatively non-specific structuresexpressed on their targets rather than more sophisticated, specificstructures which are recognised by the adaptive immune system (Hoffmannet al., 1999). Examples of cells of the innate immune system aremacrophages and dendritic cells but also granulocytes (e.g.neutrophils), natural killer cells and others. By contrast, cells of theadaptive immune system recognize specific, antigenic structures,including peptides, in the case of T cells and peptides as well asthree-dimensional structures in the case of B cells. The adaptive immunesystem is much more specific and sophisticated than the innate immunesystem and improves upon repeat exposure to a given pathogen/antigen.Phylogenetically, the innate immune system is much older and can befound already in very primitive organisms. Nevertheless, the innateimmune system is critical during the initial phase of antigenic exposuresince, in addition to containing pathogens, cells of the innate immunesystem, i.e. APCs, prime cells of the adaptive immune system and thustrigger specific immune responses leading to clearance of the intruders.In sum, cells of the innate immune system and in particular APCs play acritical role during the induction phase of immune responses by a)containing infections by means of a primitive pattern recognition systemand b) priming cells of the adaptive immune system leading to specificimmune responses and memory resulting in clearance of intrudingpathogens or of other targets (Roitt et al., 1998). These mechanisms mayalso be important to clear or contain tumor cells.

As mentioned above, cells of the innate immune system recognise patternsexpressed on their respective targets. Examples are lipopolysaccharides(LPS) in the case of Gram-negative bacteria, mycobacterial glycolipids,lipoteichoic acids of Gram-positive bacteria, mannans of yeast anddouble stranded RNAs of viruses (Hoffmann et al., 1999). In additionthey may recognise patterns such as altered glycosylations of proteinson tumor cells.

Recent findings describe DNAs of protozoan or lower eukaryotes as afurther pattern recognised by the innate (but possibly also by theadaptive) immune system of mammals (and probably most if not allvertebrates) (Krieg, 1996; Lipford et al., 1998).

The immune system recognises lower organisms including bacteria probablydue to structural and sequence usage differences between pathogen andhost DNA. In particular short stretches of DNA, derived fromnon-vertebrates or in form of short synthetic ODNs containingnonmethylated cytosine-guanine dinucleotides (CpG) in a certain basecontext, are targeted (Krieg et al., 1995). CpG motifs are found at theexpected frequency in bacterial DNA but are much less frequent invertebrate DNA (Lipford et al., 1998; Pisetsky, 1999). In addition,non-vertebrate (i.e. bacterial) CpG motifs are not methylated whereasvertebrate CpG sequences are. These differences between bacterial DNAand vertebrate DNA allow vertebrates to recognise non-vertebrate DNA asa danger signal.

Natural CpG-containing DNA, ODNs, as well as thiophosphate-substituted(exchange of thiophosphate residues for phosphate) ODNs containing CpGmotifs (CpG-ODN) are not only potent activators of immune cellproliferation and humoral immune responses (Krieg et al., 1995), butalso stimulate strong cellular immune responses (reviewed in Lipford etal., 1998). DNA/ODNs containing non-methylated CpG motifs can directlyactivate monocytic cells (dendritic cells, macrophages) and B cells.Likely, natural killer (NK) cells are not directly activated but respondto monocyte-derived IL-12 (interleukin 12) with a marked increase intheir IFN-γ production (Chace et al., 1997). In consequence, theinduction of monocytes and NK cells by CpG DNA promotes the induction ofTh1-type responses and the development of cytotoxic T cells.

Ribonucleic acid based on inosine and cytosine, likepolyinosinic-polycytidylic acid (poly I:C), is known to promoteTh1-specific immune responses. It is known to stimulate macrophages toproduce cytokines such as IL-1α and IL-12 (Manetti et al., 1995), it isalso known as a potent interferon type 1 inducer (Manetti et al., 1995)and a potent NK cell stimulator (Cavanaugh et al., 1996).

This effect, however, was strictly restricted to ribonucleic acidcontaining inosine and cytidine residues (WO98/16247).

Investigations by the inventors of the present invention showed thatODNs containing non-methylated CpG motifs, although being efficient instimulating immune system, have essential disadvantages, especially withrespect to specificity (high background) and induction of side effects,such as high systemic TNF-α generation. High systemic TNF-α release isknown to cause toxic shock syndrome, which can cause death of afflictedpatients.

It is therefore an object of the present invention to provide suitablenovel ODNs which do not have such drastic side effects as ODNs based onCpG sequences. It is a further object to reduce the side effects ofpharmaceutical compositions containing known ODNs and to provide safeand efficient well-tolerable pharmaceutical compositions with efficient,immunostimulatory properties which are suitable for vaccination ofanimals, especially of mammals, including humans.

This object is solved by immunostimulatory oligodeoxynucleic acidmolecule (ODN) having the structure according to formula (I)

any X is O or S,whereinany NMP is a 2′ deoxynucleoside monophosphate or monothiophosphate,selected from the group consisting of deoxyadenosine-, deoxyguanosine-,deoxyinosine-, deoxycytosine-, deoxyuridine-, deoxythymidine-,2-methyl-deoxyinosine-, 5-methyl-deoxycytosine-, deoxypseudouridine-,deoxyribosepurine-, 2-amino-deoxyribosepurine-, 6-S-deoxyguanine-,2-dimethyl-deoxyguanosine- or N-isopentenyl-deoxyadenosine-monophosphateor -monothiophosphate,NUC is a 2′ deoxynucleoside, selected from the group consisting ofdeoxyadenosine-, deoxyguanosine-, deoxyinosine-, deoxycytosine-,deoxyuridine-, deoxythymidine-, 2-methyl-deoxyinosine-,5-methyl-deoxycytosine-, deoxypseudouridine-, deoxyribosepurine-,2-amino-deoxyribosepurine-, 6-S-deoxyguanine-,2-dimethyl-deoxyguanosine- or N-isopentenyl-deoxyadenosine,a and b are integers from 0 to 100 with the proviso that a+b is between4 and 150,B and E are common groups for 5′ or 3′ ends of nucleic acid molecules.

Surprisingly it turned out that ODNs containing deoxyinosine residues(I-ODNs) show an immunostimulatory effect comparable or in manyinstances even better than ODNs containing CpG motifs. Moreover, ODNsaccording to the present invention produce more specific immuneresponses to a given antigen or antigen fragment than CpG ODNs. Inaddition, ODNs according to the present invention reduced the inductionof adverse side reactions, especially the induction of systemic TNF-α orIL-6.

Whereas certain immunostimulatory effects had been described for inosinecontaining RNA molecules, such as poly-IC or the molecules mentioned inWO98/16247, it surprisingly turned out that deoxynucleic acid moleculescontaining deoxyinosine residues, may be good immunostimulating ODNs.

In addition, the I-ODNs according to the present invention are—incontrast to ODNs based on the specific CpG motif—not dependent on aspecific motif or a palindromic sequence as described for the CpGoligonucleotides (see e.g. EP 0 468 520 A2, WO96/02555, WO98/18810,WO98/37919, WO98/40100, WO98/52581, WO99/51259 and WO99/56755, allincorporated herein by reference). Therefore, one group of I-ODNsaccording to the present invention may preferably contain a CI motif(and therefore ODNs described in these incorporated references, whereinone or more guanosine residues are replaced with deoxyinosine residuesare preferred embodiments of the present ODNs). It is not necessary forits principle immunostimulatory property, since I-ODNs with an Inosinenot placed in a CI or IC context exhibit immunostimulatory properties aswell.

The I-ODN according to the present invention is therefore a DNA moleculecontaining a deoxyinosine residue which is preferably provided in singlestranded form.

The I-ODN according to the present invention may be isolated throughrecombinant methods or chemically synthesized. In the latter case, theI-ODN according to the present invention may also contain modifiedoligonucleotides which may be synthesized using standard chemicaltransformations, such as methylphosphonates or other phosphorous basedmodified oligonucleotides, such as phosphotriesters, phosphoamidates andphosphorodithiorates. Other non-phosphorous based modifiedoligonucleotides can also be used (Stirchak et al., March 17 (1989),6129-6141), however, monophosphates or monothiophosphates being thepreferred 2′ deoxynucleoside monophosphate to be used in the presentinvention.

The NMPs of the I-ODNs according to the present invention are preferablyselected from the group consisting of deoxyadenosine-, deoxyguanosine-,deoxyinosine-, deoxycytosine-, deoxyuridine-, deoxythymidine-,2-methyl-deoxyinosine-, 5-methyl-deoxycytosine-monophosphate or-monothiophosphate (as usual, the phosphate or thiophosphate group is 5′of the deoxyribose). Whereas it is essential for the ODNs based on theCpG motif that this motif is unmethylated, this is surprisingly not thecase for the ODNs according to the present invention, wherein e.g.2-methyl-deoxyinosine or 5-methyl-deoxycytosine residues have no generalnegative effect on immunostimulatory properties of the ODNs according tothe present invention. Alternatively, instead of the 2-deoxy-forms ofthe NMPs, also other, especially inert, groups may be present at the2-site of the ribose group, such as e.g. —F, —NH₂, —CH₃, especially—CH₃. Of course, —OH and SH groups are excluded for the I-ODNs accordingto the present invention to be present on the 2′-site of the ribose,especially the ribose residue for the inosine NMP.

The length of the ODNs according to the present invention is in therange of the standard ODNs used according to the prior art. Thereforemolecules with a total length under 4 and above 150 show graduallydecreasing immunostimulatory potential. Preferred ODNs contain between10 and 60, especially between 15 and 40 bases (nucleosides), implyingthat a+b in formula I is between 10 and 60, preferably between 15 and 40in these preferred embodiments.

Whereas the ribonucleic acid molecules containing inosine and cytidinedescribed to be immunostimulatory in the prior art have been large andrelatively undefined polynucleic acids with molecular weights far above200,000 (a commercially available polyinosinic-polycytidylic acid fromSigma Chemicals has a molecular weight ranging from 220,000 to 460,000(at least 500-1000 C+I residues). The molecules according to the presentinvention are DNA molecules of much shorter length with a well definedlength and composition, being highly reproducible in products.

It is further preferred that the deoxyinosine containing NMP of theI-ODNs according to formula I is a monothiophosphate with one to foursulfur atoms and that also further NMPs, especially all further NMPs,are present as nucleoside monothiophosphates, because such ODNs displayhigher nuclease resistance (it is clear for the present invention thatthe “mono” in the “monothiophosphates” relates to the phosphate, i.e.that one phosphate group (one phosphor atom) is present in each NMP).Preferably, at least one of X₁ and X₂ is S and at least one of X₃ and X₄is O in the NMPs according to the present invention. Preferably, X₃ andX₄ are O. (X₃ may be (due to synthesis of the NMP) derived e.g. from thephosphate group or from the 3′-group of the NMP-ribose).

Preferably the ODNs according to the present invention contain thesequence

(SEQ ID NO: 28) hhh wdi dhh h, (SEQ ID NO: 10)nhh hhh wdi nhh hhh hhh wn, (SEQ ID NO: 11) nhh wdi din hhh hdi ndi nh,(SEQ ID NO: 12) nhh hhh wdi dhh hhh hhh wn or (SEQ ID NO: 13)nhh wdi did hhh hdi ddi dh,whereinany n is a 2′-deoxynucleoside monophosphate or monothiophosphate,selected from the group consisting of deoxyadenosine-, deoxyguanosine-,deoxycytosine- or deoxythymidine-monophosphate or -monothiophosphate,any h is a 2′-deoxynucleoside monophosphate or monothiophosphate,selected from the group consisting of deoxyadenosine-, deoxycytosine- ordeoxythymidine-monophosphate or -monothiophosphatei is deoxyinosine-monophosphate or -monothiophosphate,any w is a 2′-deoxynucleoside monophosphate or monothiophosphate,selected from the group consisting of deoxyadenosine- ordeoxythymidine-monophosphate or -monothiophosphate, andany d is a 2′-deoxynucleoside monophosphate or monothiophosphate,selected from the group consisting of deoxyadenosine-, deoxyguanosine-or deoxythymidine-monophosphate or -monothiophosphate.

As outlined above, a specific motif (such as CpG or a palindrome) is notnecessary for the I-ODNs according to the present invention. However,ODNs containing a CI motif are preferred so that in a preferredembodiment the ODN according to formula I contains

at least one 2′ deoxycytosine-monophosphate or -monothiophosphate3′-adjacent to a 2′-deoxyinosine-monophosphate or -monothiophosphate toform such a 5′-CI-3′-motif.

Preferred ODNs according to the present invention contain one or more ofthe sequence

(SEQ ID NO: 1) gacitt, (SEQ ID NO: 2) iacitt, (SEQ ID NO: 3) gaictt,(SEQ ID NO: 4) iaictt,whereina is deoxyadenosine-monophosphate or -monothiophosphate,g is deoxyguanosine-monophosphate or -monothiophosphate,i is deoxyinosine-monophosphate or -monothiophosphate,c is deoxycytosine-monophosphate or -monothiophosphate andt is deoxythymidine-monophosphate or -monothiophosphate.

The I-ODNs according to the present invention are especially suitablefor application in the pharmaceutical field, e.g. to be applied as amedicine to an animal or to humans. They are specifically adapted to actas an immunostimulatory agent, especially in or together with vaccinecompositions.

Therefore, the present invention also relates to a pharmaceuticalcomposition comprising an ODN according to the present invention.

Since a preferred pharmaceutical composition according to the presentinvention is a vaccine, this composition should contain an antigenbesides the ODN according to the present invention. The potential ofthis antigen to raise a protection/immune response of the vaccinatedindividual is strongly increased by combining it with the ODNs accordingto the present invention, especially due to their immunostimulatoryactivity.

A vaccine can contain a whole variety of different antigens. Examples ofantigens are whole-killed organisms such as inactivated viruses orbacteria, fungi, protozoa or even cancer cells. Antigens may alsoconsist of subfractions of these organisms/tissues, of proteins, or, intheir most simple form, of peptides. Antigens can also be recognised bythe immune system in form of glycosylated proteins or peptides and mayalso be or contain polysaccharides or lipids. Short peptides can be usedsince for example cytotoxic T cells (CTL) recognize antigens in form ofshort usually 8-11 amino acids long peptides in conjunction with majorhistocompatibility complex (MHC) (Rammensee et al., Immunogenetics 41,(1995), 178-228). B cells recognize longer peptides starting at around15 amino acids (Harrow et al, Cold Spring Harbor: Cold Spring HarborLaboratory, (1988)). By contrast to T cell epitopes, the threedimensional structure of B cell antigens may also be important forrecognition by antibodies. In order to obtain sustained,antigen-specific immune responses, adjuvants are helpful to triggerimmune cascades that involve all cells of the immune system necessary.Primarily, adjuvants are acting, but are not restricted in their mode ofaction, on so-called antigen presenting cells (APCs). These cellsusually first encounter the antigen(s) followed by presentation ofprocessed or unmodified antigen to immune effector. Intermediate celltypes may also be involved. Only effector cells with the appropriatespecificity are activated in a productive immune response. The adjuvantmay also locally retain antigens and co-injected other factors. Inaddition the adjuvant may act as a chemoattractant for other immunecells or may act locally and/or systemically as a stimulating agent forthe immune system.

According to a preferred embodiment, T cell epitopes are used asantigens. Alternatively, a combination of T cell epitopes and B cellepitopes may also be preferred.

The antigens to be used in the present compositions are not critical.Also mixtures of different antigens are of course possible to be usedaccording to the present invention. Preferably, proteins or peptidesderived from a viral or a bacterial pathogen or from fungi or parasitesare used as such antigens (including derivatized antigens orglycosylated or lipidated antigens or polysaccharides or lipids).Another preferred source of antigens are tumor antigens. Preferredpathogens are selected from human immunodeficiency virus (HIV),hepatitis A and B viruses, hepatitis C virus (HCV), Rous sarcoma virus(RSV), Epstein Barr virus (EBV) Influenza virus, Rotavirus,Staphylococcus aureus, Chlamydia pneumonias, Chlamydia trachomatis,Mycobacterium tuberculosis, Streptococcus pneumonias, Bacillusanthracis, Vibrio cholerae, Plasmodium sp. (Pl. falciparum, Pl. vivax,etc.), Aspergillus sp. or Candida albicans. Antigens may also bemolecules expressed by cancer cells (tumor antigens). The derivationprocess may include the purification of a specific protein from thepathogen/cancer cells, the inactivation of the pathogen as well as theproteolytic or chemical derivatization or stabilisation of such aprotein. In the same way also tumor antigens (cancer vaccines) orautoimmune antigens may be used in the pharmaceutical compositionaccording to the present invention. With such compositions a tumorvaccination or a treatment for autoimmune diseases may be performed.

In the case of peptide antigens the use of peptidemimitopes/agonists/superagonists/antagonists or peptides changed incertain positions without affecting the immunologic properties ornon-peptide mimitopes/agonists/superagonists/antagonists (reviewed inSparbier and Walden, 1999) is included in the current invention. Peptideantigens may also contain elongations either at the carboxy or at theamino terminus of the peptide antigen facilitating interaction with thepolycationic compound(s) or the immunostimulatory compound(s). For thetreatment of autoimmune diseases peptide antagonists may be applied.

Antigens may also be derivatized to include molecules enhancing antigenpresentation and targeting of antigens to antigen presenting cells.

In one embodiment of the invention the pharmaceutical composition servesto confer tolerance to proteins or protein fragments and peptides whichare involved in autoimmune diseases. Antigens used in this embodimentsserve to tolerize the immune system or downregulate immune responsesagainst epitopes involved in autoimmune processes.

Preferably the pharmaceutical composition according to the presentinvention, especially in the form of a vaccine, further comprises apolycationic polymer, preferably a polycationic peptide, especiallypolyarginine, polylysine or an antimicrobial peptide.

The polycationic compound(s) to be used according to the presentinvention may be any polycationic compound which shows thecharacteristic effect according to the WO 97/30721. Preferredpolycationic compounds are selected from basic polypeptides, organicpolycations, basic polyaminoacids or mixtures thereof. Thesepolyaminoacids should have a chain length of at least 4 amino acidresidues (see: Tuftsin as described in Goldman et al (1983)). Especiallypreferred are substances containing peptidic bounds, like polylysine,polyarginine and polypeptides containing more than 20%, especially morethan 50% of basic amino acids in a range of more than 8, especially morethan 20, amino acid residues or mixtures thereof. Other preferredpolycations and their pharmaceutical compositions are described in WO97/30721 (e.g. polyethyleneimine) and WO 99/38528. Preferably thesepolypeptides contain between 20 and 500 amino acid residues, especiallybetween 30 and 200 residues.

These polycationic compounds may be produced chemically or recombinantlyor may be derived from natural sources.

Cationic (poly)peptides may also be polycationic anti-bacterialmicrobial peptides with properties as reviewed in (Ganz and Lehrer,1999; Hancock, 1999). These (poly)peptides may be of prokaryotic oranimal or plant origin or may be produced chemically or recombinantly(Andreu and Rivas, 1998; Ganz and Lehrer, 1999; Simmaco et al., 1998).Peptides may also belong to the class of defensins (Ganz, 1999; Ganz andLehrer, 1999).

Such host defense peptides or defensives are also a preferred form ofthe polycationic polymer according to the present invention. Generally,a compound allowing as an end product activation (or down-regulation) ofthe adaptive immune system, preferably mediated by APCs (includingdendritic cells) is used as polycationic polymer.

Especially preferred for use as polycationic substance in the presentinvention are cathelicidin derived antimicrobial peptides or derivativesthereof (A 1416/2000, incorporated herein by reference), especiallyantimicrobial peptides derived from mammal cathelicidin, preferably fromhuman, bovine or mouse, or neuroactive compounds, such as (human) growthhormone.

Polycationic compounds derived from natural sources include HIV-REV orHIV-TAT (derived cationic peptides, antennapedia peptides, chitosan orother derivatives of chitin) or other peptides derived from thesepeptides or proteins by biochemical or recombinant production. Otherpreferred polycationic compounds are cathelin or related or derivedsubstances from cathelin. For example, mouse cathelin is a peptide whichhas the amino acid sequenceNH₂-RLAGLLRKGGEKIGEKLKKIGOKIKNFFQKLVPQPE-COOH (SEQ ID NO:5). Related orderived cathelin substances contain the whole or parts of the cathelinsequence with at least 15-20 amino acid residues. Derivations mayinclude the substitution or modification of the natural amino acids byamino acids which are not among the 20 standard amino acids. Moreover,further cationic residues may be introduced into such cathelinmolecules. These cathelin molecules are preferred to be combined withthe antigen and the immunogenic ODN according to the present invention.However, these cathelin molecules surprisingly have turned out to bealso effective as an adjuvant for a antigen without the addition offurther adjuvants. It is therefore possible to use such cathelinmolecules as efficient adjuvants in vaccine formulations with or withoutfurther immunactivating substances.

Another preferred polycationic substance to be used according to thepresent invention is a synthetic peptide containing at least 2KLK-motifs separated by a linker of 3 to 7 hydrophobic amino acids (A1789/2000, incorporated herein by reference).

It was very surprising that the immunostimulating effect of thepharmaceutical composition according to the present invention wassignificantly higher than it could be expected from the addition of theeffects of each single component or even the addition of the effects ofthe ODN or the polycation with the antigen.

B and E in formula I are common groups for 5′ and/or 3′ ends of nucleicacid molecules. Examples for such groups are readily available for theskilled man in the art (see e.g. “Oligonucleotides and Analogues—APractical Approach” (1991), ed. Eckstein, Oxford University Press). Forthe I-ODNs according to the present invention B and/or E are preferablyselected independently from —H, —CH₃, —COCH₃, —OH, —CHO, a phosphate,thiophosphate, sulfate or a thiosulfate group, or a phosphoalkylgroup,especially with an alkyl length of C₁-C₆ and/or with a terminal aminogroup (the amino group may e.g. be used for further labelling of theI-ODNs according to the present invention, e.g. —PO₄—(CH₂)_(n)—NH₂ or—PO₄—(CH₂)_(n)—NH-Label). Especially preferred as B are nucleosides,especially the 2′ deoxynucleotides mentioned above (i.e. without thephosphate or thiophosphate group). Alternatively these groups may alsocontain linker groups to other molecules, especially carrier moleculesor labels. In such forms of ODNs wherein the ODNs are bound to solidsurfaces or particles or labels, these surfaces, particles, labels, etc.are then also part of the B and/or E groups.

Of course, any ionised (salt) form or tautomeric forms of the moleculesaccording to formula I are included in this formula I.

The pharmaceutical composition according to the present invention mayfurther comprise further active ingredients (pharmaceutically activesubstances), especially substances which are usable in a vaccineconnection. Preferred embodiments of such further active ingredients arecytokines, antiinflammatory substances, antimicrobial substances orcombinations thereof.

Of course, the pharmaceutical composition according to the presentinvention may further contain auxiliary substances, especially apharmaceutically acceptable carrier, buffer substances, stabilizers orcombinations thereof.

The relative amounts of the ingredients in the present pharmaceuticalcomposition are highly dependent on the necessities of the individualantigen and on the animal/human to which this composition should beapplied to. Therefore, the pharmaceutical composition according to thepresent invention preferably contains one or more ODNs according to thepresent invention, preferably 1 pg to 10 g, preferably 1 ng to 1 g, morepreferred 100 ng to 10 mg, especially 10 mg to 1 mg. The antigen as wellas the polycationic polymer may be applied in similar dosages, a rangeof 1 to 10,000 mg antigen and 0.1 to 1,000 mg polycation per vaccinationis preferred.

The present compositions may be applied to a patient, e.g. a vaccinationcandidate, in efficient amounts e.g. by weekly, bi-weekly or monthlyintervals. Patients to be treated with the present compositions may alsobe vaccinated repeatedly or only once. A preferred use of the presentinvention is the active immunisation, especially of humans or animalswithout protection against the specific antigen.

The route of application for the present composition is not critical,e.g. subcutaneous, intramuscular, intradermal or transdermal injectionis suitable as well as oral uptake.

It is also possible to apply the present composition separately e.g. byinjecting the immunostimulating substance separately from theantigen/polycation composition. The present invention is therefore alsodirected to a kit comprising a composition containing the antigen andthe polycationic polymer as one component and a composition containingthe immunostimulating or chemotactic substance as a second component.

The components may be applied at the same site or time, however, anapplication at different sites or at a different time or for a differenttime period is also possible. It is also possible to vary the systemicor local applications of the composition or the components,respectively.

Details of the present invention are described by the following examplesand the figures, but the invention is of course not limited thereto.

FIG. 1 shows the immune response against the ovalbumin-derived peptideOVA₂₅₇₋₂₆₄ after the injection of OVA₂₅₇₋₂₆₄, poly-L-arginine (pR 60)and deoxyinosine I-containing oligodeoxynucleotides (I-ODN) or CpG 1668.Mice were injected into the hind footpads with mixtures as indicated.Four days later draining lymph node cells were ex vivo stimulated withOVA₂₅₇₋₂₆₄. The number of IFN-g-producing cells was determined 24 hourslater using an ELISPOT assay. Results are expressed as the number ofspots/1×10⁶ lymph node cells.

FIG. 2 shows the induction of systemic TNF-a production after theinjection of OVA₂₅₇₋₂₆₄, poly-L-arginine (pR 60) and I-containingoligodeoxynucleotides (I-ODN) or CpG 1668. Mice were injected into thehind footpads with mixtures as indicated. One hour after injection bloodwas taken from the tail vein and serum was prepared. The concentrationof TNF-a in the sera was determined using an ELISA.

FIG. 3 shows the immune response against the Ovalbumin-derived peptideOVA₂₅₇₋₂₆₄ after the injection of OVA₂₅₇₋₂₆₄, poly-L-arginine (pR60) anddeoxyinosine-containing oligodeoxynucleotides (I-ODN), CpG 1668 or GpC.Mice were injected into the hind footpads with mixtures as indicated.Four days later, draining lymph node cells were ex vivo stimulated withOVA₂₅₇-264, an irrelevant peptide mTRP2₁₈₁₋₁₈₈ (murine tyrosinaserelated protein-2, VYDFFVWL (SEQ ID NO: 8)) or pR 60. The number ofIFN-g producing cells was determined 24 hours later using an ELISPOTassay. Results are expressed as the number of spots/1×10⁶ lymph nodecells with standard deviation of triplicates.

FIG. 4 shows the induction of systemic TNF-a production after theinjection of OVA₂₅₇₋₂₆₄, poly-L-arginine (pR 60) and I-containingoligodeoxynucleotides (I-ODN), GpC or CpG 1668. Mice were injected intothe hind footpads with mixtures as indicated. One hour after injectionblood was taken from the tail vein and serum was prepared. Theconcentration of TNF-a and IL-6 in the sera was determined usingcytokine-specific ELISAs.

FIG. 5 shows the immune response against the Ovalbumin-derived peptideOVA₂₅₇₋₂₆₄ after the injection of TRP-2, poly-L-arginine, CpG 1668 orrandom 20-mer sequences containing deoxyinosine. Mice were injected intothe hind footpads with mixtures as indicated. Four days later, draininglymph node cells were ex vivo stimulated with TRP-2, an irrelevantpeptide OVA₂₅₇₋₂₆₄ or pR 60. The number of IFN-g producing cells wasdetermined 24 hours later using an ELISPOT assay. Results are expressedas the number of spots/1×10⁶ lymph node cells with standard deviation oftriplicates.

FIG. 6 shows the combined injection of I-ODN and poly-L-arginine (pR 60)together with a Melanoma-derived peptide.

FIG. 7 shows that the combined injection of I-ODN and pR 60 togetherwith a Melanoma-derived peptide reduces the induction of systemic TNF-αand IL-6.

FIG. 8 shows the combined injection of a random 10-mer I-ODN and pR 60together with a Melanoma-derived peptide.

FIG. 9 shows that the combined application of ovalbumin (OVA) witholigo-dIC_(26-mer) and pR enhances production of OVA-specific IgGantibodies. Mice were injected subcutaneously into the footpad withmixtures as indicated. At day 24 and 115 after injection, sera werecollected and screened by ELISA for OVA-specific IgG2a (A) and IgG1 (B)antibodies. The results are shown as the antibody titer.

EXAMPLES

In all experiments thiophosphate-substituted ODNs (with thiophosphateresidues substituting for phosphate, hereafter called “thiophosphatesubstituted oligodeoxynucleotides”) were used since such ODNs displayhigher nuclease resistance (Ballas et al., 1996; Krieg et al., 1995;Parronchi et al., 1999).

Example 1 The Combined Injection of Different I-ODNs and Poly-L-Arginine(pR 60) Synergistically Enhances the Immune Response Against anOvalbumin-Derived Peptide

-   Mice C57BI/6 (Harlan/Olac)-   Peptide OVA₂₅₇₋₂₆₄-Peptide (SIINFEKL) (SEQ ID NO:6), a MHC class I    (H-2 Kb)-restricted epitope of chicken ovalbumin (Rotzschke et al.,    1991), was synthesized using standard solid phase F-moc chemistry    synthesis, HPLC purified and analysed by mass spectroscopy for    purity.    -   Dose: 300 mg/mouse-   Poly-L-arginine-60 (pR60) Poly-L-arginine with an average degree of    polymerization of 60 arginine residues; SIGMA chemicals    -   Dose: 100 mg/mouse-   CpG-ODN 1668 thiophosphate substituted ODNs containing a CpG motif:    -   tcc atg acg ttc ctg atg ct (SEQ ID NO:7), were synthesized by        NAPS GmbH, Göttingen.    -   Dose: 5 nmol/mouse-   I-ODN 1 thiophosphate substituted ODNs containing deoxyinosine:    -   tcc ati aci ttc ctg atg ct (SEQ ID NO:14), were synthesized by        NAPS GmbH, Gottingen.    -   Dose: 5 nmol/mouse-   I-ODN 2 thiophosphate substituted ODNs containing deoxyinosine:    -   tcc atg aci ttc ctg atg ct (SEQ ID NO:15), were synthesized by        NAPS GmbH, Göttingen.    -   Dose: 5 nmol/mouse-   I-ODN 3 thiophosphate substituted ODNs containing deoxyinosine:    -   tcc ati aci ttc cti ati ct (SEQ ID NO:16), were synthesized by        NAPS GmbH, Gottingen.    -   Dose: 5 nmol/mouse        Experimental Groups (5 Mice Per Group)    -   1. OVA₂₅₇₋₂₆₄    -   2. OVA₂₅₇₋₂₆₄+pR 60    -   3. OVA₂₅₇₋₂₆₄+CpG 1668    -   4. OVA₂₅₇₋₂₆₄+I-ODN 1    -   5. OVA₂₅₇₋₂₆₄+I-ODN 2    -   6. OVA₂₅₇₋₂₆₄+I-ODN 3    -   7. OVA₂₅₇₋₂₆₄+CpG 1668+pR 60    -   8. OVA₂₅₇₋₂₆₄+I-ODN 1+pR 60    -   9. OVA₂₅₇₋₂₆₄+I-ODN 2+pR 60    -   10. OVA₂₅₇₋₂₆₄+I-ODN 3+pR 60

On day 0 mice were injected into each hind footpad with a total volumeof 100 ml (50 ml per footpad) containing the above mentioned compounds.Animals were sacrificed 4 days after injection and popliteal lymph nodeswere harvested. Lymph nodes were passed through a 70 mm cell strainerand washed twice with DMEM medium (GIBCO BRL) containing 5% fetal calfserum (FCS, SIGMA chemicals). Cells were adjusted to 3×10⁶ cells/ml inDMEM/5%/FCS. An IFN-g ELISPOT assay was carried out in triplicates asdescribed (Miyahira et al., 1995). This method is a widely usedprocedure allowing the quantification of antigen-specific T cells.Lymphocytes were stimulated ex vivo with medium background-control,OVA₂₅₇₋₂₆₄-peptide or Concanavalin A (Con A). Spots representing singleIFN-g producing T cells were counted and the number of background spotswas subtracted from all samples. The high number of spots detected afterthe stimulation with Con A (data not shown) indicate a good condition ofthe used lymphocytes. For each experimental group of mice the number ofspots/1×10⁶ cells are illustrated in FIG. 1.

One hour after injection blood was taken from the tail vein and serumwas prepared to determine the induction of systemic TNF-a using an ELISA(FIG. 2).

Example 2 The Exchange of Guanosine by Desoxy-Inosine Converts theNon-Immunogeneic GpC-Sequence to a Highly Immunogeneic One, Especiallywhen Combined with Poly-L-Arginine (pR60)

-   Mice C57Bl/6 (Harlan/Olac)-   Peptide OVA₂₅₇₋₂₆₄-Peptide (SIINFEKL (SEQ ID NO:6)), a MHC class I    -   (H-2 Kb)-restricted epitope of chicken ovalbumin (Rotzschke et        al., 1991), was synthesized using standard solid phase F-moc        synthesis, HPLC purified and analysed by mass spectroscopy for        purity.    -   Dose: 300 μg/mouse-   Poly-L-arginine 60 (pR60) Poly-L-arginine with an average degree of    polymerization of 60 arginine residues; SIGMA chemicals    -   Dose: 100 μg/mouse-   CpG-ODN 1668 thiophosphate substituted ODNs containing a CpG motif:    tcc atg acg ttc ctg atg ct (SEQ ID NO:7), were synthesized by NAPS    GmbH, Göttingen.    -   Dose: 5 nmol/mouse-   GpC-ODN thiophosphate substituted ODNs containing an    non-immunogeneic GpC motif: tcc atg agc ttc ctg atg ct (SEQ ID    NO:17) were synthesized by NAPS GmbH, Göttingen.    -   Dose: 5 nmol/mouse-   I-ODN 9 thiophosphate substituted ODNs containing deoxyinosine: tcc    atg aic ttc ctg atg ct (SEQ ID NO:18) were synthesized by NAPS GmbH,    Göttingen.    -   Dose: 5 nmol/mouse-   I-ODN 10 thiophosphate substituted ODNs containing deoxyinosine: tcc    ati aic ttc cti ati ct (SEQ ID NO:19) were synthesized by NAPS GmbH,    Göttingen.    -   Dose: 5 nmol/mouse        Experimental Groups (5 Mice Per Group)-   OVA₂₅₇₋₂₆₄-   OVA₂₅₇₋₂₆₄+pR 60-   OVA₂₅₇₋₂₆₄+CpG 1668-   OVA₂₅₇₋₂₆₄+GpC-   OVA₂₅₇₋₂₆₄+I-ODN 9-   OVA₂₅₇₋₂₆₄+I-ODN 10-   OVA₂₅₇₋₂₆₄+CpG 1668+pR 60-   OVA₂₅₇₋₂₆₄+GpC+pR 60-   OVA₂₅₇₋₂₆₄+I-ODN 9+pR 60-   OVA₂₅₇₋₂₆₄+I-ODN 10+pR 60

On day 0 mice were injected into each hind footpad with a total volumeof 100 μl (50 μl per footpad) containing the above mentioned compounds.Animals were sacrificed 4 days after injection and popliteal lymph nodeswere harvested. Lymph nodes were passed through a 70 μm cell strainerand washed twice with DMEM medium (GIBCO BRL) containing 5% fetal calfserum (FCS, SIGMA chemicals). Cells were adjusted to 3×10⁶ cells/ml inDMEM/5% FCS. An IFN-g ELISPOT assay was carried out in triplicates asdescribed (Miyahira et al., 1995). This method is a widely usedprocedure allowing the quantification of antigen-specific T cells.Lymphocytes were stimulated ex vivo in triplicates with medium(background), OVA₂₅₇₋₂₆₄-peptide, an irrelevant peptide mTRP-2₁₈₁₋₁₈₈(murine tyrosinase related protein-2, VYDFFVWL (SEQ ID NO:8)), pR 60 andConcanavalin A (Con A). Spots representing single IFN-g producing Tcells were counted and the number of background spots was subtractedfrom all samples. The high number of spots detected after thestimulation with Con A (data not shown) indicate a good condition of theused lymphocytes. For each experimental group of mice the number ofspots/1×10⁶ cells are illustrated in FIG. 3, the standard deviation ofex vivo-stimulated triplicates are given. One hour after injection bloodwas taken from the tail vein and serum was prepared to determine theinduction of systemic TNF-a and IL-6 using cytokine-specific ELISAs(FIG. 4).

Example 3 The Combined Injection of Random 20-Mer Sequences ContainingDeoxyinosine and a Melanoma-Derived Peptide Induces a Strong ImmuneResponse Against the Peptide which can be Further Enhanced by theCo-Application of Poly-L-Arginine (pR 60)

-   Mice C57Bl/6 (Harlan/Olac)-   Peptide TRP-2-peptide (VYDFFVWL (SEQ ID NO:8), a MHC class I    (H-2K^(b))-restricted epitope of mouse tyrosinase related protein-2    (Bloom et al., 1997) was synthesized by standard solid phase F-moc    synthesis, HPLC purified and analyzed by mass spectroscopy for    purity.    -   Dose: 300 μg/mouse-   Poly-L-arginine 60 (pR60) Poly-L-arginine with an average degree of    polymerization of 60 arginine residues; SIGMA chemicals    -   Dose: 100 μg/mouse-   CpG-ODN 1668 thiophosphate substituted ODNs containing a CpG motif:    tcc atg acg ttc ctg atg ct (SEQ ID NO:7), were synthesized by NAPS    GmbH, Göttingen.    -   Dose: 5 nmol/mouse-   Wdi thiophosphate substituted ODNs:    -   nhh hhh wdi nhh hhh hhh wn (SEQ ID NO:10) were synthesized by        NAPS GmbH, Göttingen.    -   Dose: 5 nmol/mouse-   Wdidin thiophosphate substituted ODNs:    -   nhh hhh wdi nhh hhh hhh wn (SEQ ID NO:10) were synthesized by        NAPS GmbH, Göttingen.    -   Dose: 5 nmol/mouse-   Wdid thiophosphate substituted ODNs:    -   nhh hhh wdi dhh hhh hhh wn (SEQ ID NO:12) were synthesized by        NAPS GmbH, Göttingen.    -   Dose: 5 nmol/mouse-   Wdidid thiophosphate substituted ODNs:    -   nhh wdi did hhh hdi ddi dh (SEQ ID NO:13) were synthesized by        NAPS GmbH, Göttingen.    -   Dose: 5 nmol/mouse        Experimental Groups (5 Mice Per Group)        1. TRP-2        2. TRP-2+pR 60        3. TRP-2+CpG 1668        4. TRP-2+wdi        5. TRP-2+wdidin        6. TRP-2+wdid        7. TRP-2+wdidid        8. TRP-2+CpG 1668+pR 60        9. TRP-2+wdi+pR 60        10. TRP-2+wdidin+pR 60        11. TRP-2+wdid+pR 60        12. TRP-2+wdidid+pR 60

On day 0 mice were injected into each hind footpad with a total volumeof 100 μl (50 μl per footpad) containing the above mentioned compounds.Animals were sacrificed 4 days after injection and popliteal lymph nodeswere harvested. Lymph nodes were passed through a 70 μm cell strainerand washed twice with DMEM medium (GIBCO BRL) containing 5% fetal calfserum (FCS, SIGMA chemicals). Cells were adjusted to 3×10⁶ cells/ml inDMEM/5% FCS. An IFN-g ELISPOT assay was carried out in triplicates asdescribed (Miyahira et al., 1995). This method is a widely usedprocedure allowing the quantification of antigen-specific T cells.Lymphocytes were stimulated ex vivo in triplicates with medium(background), TRP-2-peptide, an irrelevant OVA₂₅₇₋₂₆₄-peptide, pR 60 andConcanavalin A (Con A). Spots representing single IFN-g producing Tcells were counted and the number of background spots was subtractedfrom all samples. The high number of spots detected after thestimulation with Con A (data not shown) indicate a good condition of theused lymphocytes. For each experimental group of mice the number ofspots/1×10⁶ cells are illustrated in FIG. 5, the standard deviation ofex vivo-stimulated triplicates are given.

Example 4 The Combined Injection of I-ODN and Poly-L-Arginine (pR 60)Synergistically Enhances the Immune Response Against a Melanoma-DerivedPeptide

Experimental Groups (5 Mice Per Group)

1. TRP-2₁₈₁₋₁₈₈

2. TRP-2₁₈₁₋₁₈₈+pR 60

3. TRP-2₁₈₁₋₁₈₈+CpG 1668

4. TRP-2₁₈₁₋₁₈₈+I-ODN 2

5. TRP-2₁₈₁₋₁₈₈+CpG 1668+pR 60

6. TRP-2₁₈₁₋₁₈₈+I-ODN 2+pR 60

On day 0 mice were injected into each hind footpad with a total volumeof 100 μl (50 μl per footpad) containing the above mentioned compounds.Animals were sacrificed 4 days after injection and popliteal lymph nodeswere harvested. Lymph nodes were passed through a 70 μm cell strainerand washed twice with DMEM medium (GIBCO BRL) containing 5% fetal calfserum (FCS, SIGMA chemicals). Cells were adjusted to 3×10⁶ cells/ml inDMEM/5%/FCS. An IFN-γ ELISPOT assay was carried out in triplicates asdescribed (Miyahira et al., 1995). This method is a widely usedprocedure allowing the quantification of antigen-specific T cells.Lymphocytes were stimulated ex vivo in triplicates with mediumbackground-control, TRP-2₁₈₁₋₁₈₈-peptide, an irrelevantOVA₂₅₇₋₂₆₄-peptide and Concanavalin A (Con A). Spots representing singleIFN-γ producing T cells were counted and the number of background spotswas subtracted from all samples. The high number of spots detected afterthe stimulation with Con A (data not shown) indicate a good condition ofthe used lymphocytes. For each experimental group of mice the number ofspots/1×10⁶ cells are illustrated in FIG. 6, the standard deviation ofex vivo-stimulated triplicates are given.

One hour after injection blood was taken from the tail vein and serumwas prepared to determine the induction of systemic TNF-α (and IL-6using specific ELISAs (FIG. 7).

Example 5 The Combined Injection of Random 10-Mer I-ODN andPoly-L-Arginine (pR 60) Synergistically Enhances the Immune ResponseAgainst a Melanoma-Derived Peptide

Experimental Groups (5 Mice Per Group)

1. TRP-2₁₈₁₋₁₈₈

2. TRP-2₁₈₁₋₁₈₈+pR 60

3. TRP-2₁₈₁₋₁₈₈+CpG 1668

4. TRP-2₁₈₁₋₁₈₈+ODN 17

5. TRP-2₁₈₁₋₁₈₈+CpG 1668+pR 60

6. TRP-2₁₈₁₋₁₈₈+ODN 17+pR 60

On day 0 mice were injected into each hind footpad with a total volumeof 100 μl (50 μl per footpad) containing the above mentioned compounds.Animals were sacrificed 4 days after injection and popliteal lymph nodeswere harvested. Lymph nodes were passed through a 70 μm cell strainerand washed twice with DMEM medium (GIBCO BRL) containing 5% fetal calfserum

(FCS, SIGMA chemicals). Cells were adjusted to 3×10⁶ cells/ml inDMEM/5%/FCS. An IFN-γ ELISPOT assay was carried out in triplicates asdescribed (Miyahira et al., 1995). This method is a widely usedprocedure allowing the quantification of antigen-specific T cells.Lymphocytes were stimulated ex vivo in triplicates with mediumbackground-control, TRP-2₁₈₁₋₁₈₈-peptide, an irrelevantOVA₂₅₇₋₂₆₄-peptide and Concanavalin A (Con A). Spots representing singleIFN-γ producing T cells were counted and the number of background spotswas subtracted from all samples. The high number of spots detected afterthe stimulation with Con A (data not shown) indicate a good condition ofthe used lymphocytes. For each experimental group of mice the number ofspots/1×10⁶ cells are illustrated in FIG. 8, the standard deviation ofex vivo-stimulated triplicates are given.

-   Mice C57Bl/6 (Harlan/Olac)-   Peptide TRP-2-peptide (VYDFFVWL (SEQ ID NO: 8)), a MHC class I    (H-2K^(b))-restricted epitope of mouse tyrosinase related protein-2    (Bloom et al., 1997) was synthesized by standard solid phase F-moc    synthesis, HPLC purified and analyzed by mass spectroscopy for    purity.    -   Dose: 100 μg/mouse-   Poly-L-arginine-60 (pR60) Poly-L-arginine with an average degree of    polymerization of 60 arginine residues;    -   SIGMA chemicals    -   Dose: 100 μg/mouse-   CpG-ODN 1668 thiophosphate substituted ODNs containing a CpG motif:    tcc atg acg ttc ctg atg ct (SEQ ID NO:7), were synthesized by NAPS    GmbH, Göttingen.    -   Dose: 5 nmol/mouse-   ODN 17 thiophosphate substituted ODNs containing deoxyinosine: hhh    wdi dhh h (SEQ ID NO: 29), were synthesized by NAPS GmbH, Göttingen.    -   (h=CAT, w=AT, d=GAT)    -   Dose: 10 nmol/mouse-   Mice C57Bl/6 (Harlan/Olac) Peptide TRP-2-peptide (VYDFFVWL (SEQ ID    NO: 8)), a MHC class I (H-2K^(b))-restricted epitope of mouse    tyrosinase related protein-2 (Bloom et al., 1997) was synthesized by    standard solid phase F-moc synthesis, HPLC purified and analyzed by    mass spectroscopy for purity.    -   Dose: 100 μg/mouse-   Poly-L-arginine-60 (pR60) Poly-L-arginine with an average degree of    polymerization of 60 arginine residues; SIGMA chemicals    -   Dose: 100 μg/mouse-   CpG-ODN 1668 thiophosphate substituted ODNs containing a CpG motif:    tcc atg acg ttc ctg atg ct (SEQ ID NO:7), were synthesized by NAPS    GmbH, Göttingen.    -   Dose: 5 nmol/mouse-   I-ODN 2 thiophosphate substituted ODNs containing deoxyinosine: tcc    atg aci ttc ctg atg ct (SEQ ID NO: 15), were synthesized by NAPS    GmbH, Göttingen.    -   Dose: 5 nmol/mouse

Example 6 The Combined Application of Oligo-deoxyIC_(26-mer) andPoly-L-Arginine (pR) Enhances the Ovalbumin (OVA)-Specific HumoralResponse

-   Mice C57Bl/6 (Harlan/Olac)-   Ovalbumin (OVA) Ovalbumin from chicken egg, grade V, SIGMA    Chemicals, A-5503, Lot 54H7070    -   Dose: 50 μg/mouse-   Poly-L-arginine (pR) Poly-L-arginine with an average degree of    polymerization of 60 arginine residues; SIGMA Chemicals, P-4663, Lot    68H5903    -   Dose: 100 μg/mouse-   Oligo-deoxy IC, 26-mer oligo-dIC_(26-mer) (SEQ ID NO: 30) was    (oligo-dIC_(26-mer)) synthesized by standard phosphoamidide    chemistry on a 4 μmol scale and purified by HPLC (NAPS Göttingen,    Germany)    -   Dose: 5 nmol/mouse        Experimental Groups (4 Mice Per Group)        1. OVA+oligo-dIC_(26-mer)+pR        2. OVA+oligo-dIC_(26-mer)        3. OVA+pR        4. OVA

On day 0, mice were injected into each hind footpad with a total volumeof 100 μl (50 μl per footpad) containing the above listed compounds. Onday 24 after injection, serum was collected and screened by ELISA forthe presence of OVA-specific antibodies. These results show that theinjection of OVA in combination with oligo-dIC and pR enhanced theproduction of OVA-specific IgG antibodies when compared with injectionof OVA with each of the substances alone (FIGS. 9A, 9B). Interestingly,titers of both IgG2a and IgG1 were increased upon one single injectionof OVA with oligo-dIC/pR, implying that both Th1 and Th2 cells wereinvolved. However, after 115 days only the increased IgG2a levels werestill detectable in sera of mice injected with OVA and oligo-dIC/pR.

These data demonstrate that the combined injection of OVA with oligo-dICand pR enhances the OVA-specific humoral response. This response ischaracterized by the production of both Th1- and Th2-induced antibodyisotypes in the early phase, but later, mainly by Th1-inducedantibodies.

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The invention claimed is:
 1. A method of production of a pharmaceuticalcomposition comprising combining at least one antigen and at least oneimmunostimulatory oligodeoxynucleotide molecule (ODN) consisting of thesequence oligo-dIC_(26-mer) (SEQ ID NO:30), wherein the pharmaceuticalcomposition is further defined as comprising 10 ng to 1 mg of the ODN.2. The method of production according to claim 1, wherein thepharmaceutical composition further comprises at least one of apolycationic polymer, an antimicrobial peptide, a growth hormone, acytokine, an anti-inflammatory substance, a pharmaceutically acceptablecarrier, a buffer substance or a stabilizer.
 3. The method of productionaccording to claim 1, wherein the at least one antigen is derived fromviral or bacterial pathogens, from fungi or parasites, and/or whereinthe antigens are tumor antigens or autoimmune disease antigens.
 4. Themethod of production according to claim 2, wherein the polycationicpolymer is a synthetic peptide comprising two KLK-motifs separated by alinker of 3 to 7 hydrophobic amino acids.